A Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) for ovarian cancer.

BACKGROUND: Pronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC). METHODS: pAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC). RESULTS: pAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals. CONCLUSION: pAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer.

The First-In-Class Anti-AXL×CD3ε Pronectin™-Based Bispecific T-Cell Engager Is Active in Preclinical Models of Human Soft Tissue and Bone Sarcomas.

Sarcomas are heterogeneous malignancies with limited therapeutic options and a poor prognosis. We developed an innovative immunotherapeutic agent, a first-in-class Pronectin™-based Bispecific T-Cell Engager (pAXL×CD3ε), for the targeting of AXL, a TAM family tyrosine kinase receptor highly expressed in sarcomas. AXL expression was first analyzed by flow cytometry, qRT-PCR, and Western blot on a panel of sarcoma cell lines. The T-cell-mediated pAXL×CD3ε cytotoxicity against sarcoma cells was investigated by flow cytometry, luminescence assay, and fluorescent microscopy imaging. The activation and degranulation of T cells induced by pAXL×CD3ε were evaluated by flow cytometry. The antitumor activity induced by pAXL×CD3ε in combination with trabectedin was also investigated. In vivo activity studies of pAXL×CD3ε were performed in immunocompromised mice (NSG), engrafted with human sarcoma cells and reconstituted with human peripheral blood mononuclear cells from healthy donors. Most sarcoma cells showed high expression of AXL. pAXL×CD3ε triggered T-lymphocyte activation and induced dose-dependent T-cell-mediated cytotoxicity. The combination of pAXL×CD3ε with trabectedin increased cytotoxicity. pAXL×CD3ε inhibited the in vivo growth of human sarcoma xenografts, increasing the survival of treated mice. Our data demonstrate the antitumor efficacy of pAXL×CD3ε against sarcoma cells, providing a translational framework for the clinical development of pAXL×CD3ε in the treatment of human sarcomas, aggressive and still-incurable malignancies.

AXL-Receptor Targeted 14FN3 Based Single Domain Proteins (Pronectins™) from 3 Synthetic Human Libraries as Components for Exploring Novel Bispecific Constructs against Solid Tumors.

A highly specific AXL-receptor targeted family of non-immunoglobulin, single domain protein binders (Pronectins™) have been isolated from three (3) synthetic libraries that employ the human scaffold of the 14th domain of Fibronectin III (14FN3) and evolutionary CDRs diversity of over 25 billion loop sequences. The three libraries, each containing diversity in two loops, were designed to expand upon a human database of more than 6000 natural scaffold sequences and approximately 3000 human loop sequences. We used a bioinformatic-based approach to maximize "human" amino acid loop diversity and minimize or prevent altogether CDR immunogenicity created by the use of mutagenesis processes to generate diversity. A combination of phage display and yeast display was used to isolate 59 AXL receptor targeted Pronectins with KD ranging between 2 and 100 nM. FACS analysis with tumor cells over-expressing AXL and the use of an AXL knock-out cell line allowed us to identify Pronectin candidates with exquisite specificity for AXL receptor. Based upon several in vitro cell-based tests, we selected the best candidate, AXL54, to further characterize its in vitro cancer cells killing activity. Finally, AXL54 was used to produce the first bi-specific T cell engager protein (AXL54 [Pronectin]-linker-scFV CD3), a "new in class" protein for further testing of its anti-tumor activity in vitro and in vivo.

Engineering highly thermostable xylanase variants using an enhanced combinatorial library method.

A new directed evolution method was used to enhance the thermostability of the wild-type GH11 xylanase 2 (known as BD-11) from Hypocrea jecorina (Trichoderma reesei). Both Look-Through Mutagenesis (LTM™), which is a method for rapidly screening selected positions in the protein sequence for amino acids that introduce favorable properties, and Combinatorial Beneficial Mutagenesis (CBM™), which is a method for identifying the best ensemble of individual mutations, were employed to enhance the stability of an enzyme that has been thoroughly engineered by various means during the past 20 years. A diverse set of novel mutations was discovered, including N71D, Y73G, T95G and Y96Q. When these mutations were combined into a single construct (Hjx-81), the purified protein was active even after heating at 100°C for 20 min. This time-effective method should be generally applicable for quickly improving the physico-chemical properties of other industrial and therapeutic enzymes in only several months time.

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii.

Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear whether this is because of few attempts or of limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, >50% expressed at levels sufficient for commercial production. Three expressed at 2%-3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity achieved demonstrate the utility of Chlamydomonas reinhardtii as a robust platform for human therapeutic protein production.

Investigation of the biophysical and cell biological properties of ferroportin, a multipass integral membrane protein iron exporter.

Ferroportin is a multipass membrane protein that serves as an iron exporter in many vertebrate cell types. Ferroportin-mediated iron export is controlled by the hormone hepcidin, which binds ferroportin, causing its internalization and degradation. Mutations in ferroportin cause a form of the iron overload hereditary disease hemochromatosis. Relatively little is known about ferroportin's properties or the mechanism by which mutations cause disease. In this study, we expressed and purified human ferroportin to characterize its biochemical/biophysical properties in solution and conducted cell biological studies in mammalian cells. We found that purified detergent-solubilized ferroportin is a well-folded monomer that binds hepcidin. In cell membranes, the N- and C-termini were both cytosolic, implying an even number of transmembrane regions, and ferroportin was mainly localized to the plasma membrane. Hepcidin addition resulted in a redistribution of ferroportin to intracellular compartments that labeled with early endosomal and lysosomal, but not Golgi, markers and that trafficked along microtubules. An analysis of 16 disease-related ferroportin mutants revealed that all were expressed and trafficked to the plasma membrane but that some were resistant to hepcidin-induced internalization. The characterizations reported here form a basis upon which models for ferroportin's role in regulating iron homeostasis in health and disease can be interpreted.

Hybrid yeast-bacteria cloning system used to capture and modify adenoviral and nonviral genomes.

Adenoviral vectors are widely used to express transgenes in vitro and in vivo. A major obstacle to the generation of adenoviral vectors is the manipulation of the large (35 kb) adenoviral genome. We developed a hybrid yeast-bacteria cloning system for the creation of novel adenoviral vectors. The adenovirus 5 (Ad5) genome was cloned into a shuttle vector that contains both yeast and bacterial elements for replication and therefore functions as both a yeast artificial plasmid (YAP) and as a plasmid artificial chromosome (PAC). Any sequence can be introduced into any region of the adenoviral genome via the highly efficient homologous recombination in yeast and then these recombinants are rapidly amplified in bacteria. Adenoviral vectors are generated by introduction of the PAC into the appropriate complementing mammalian cell without the need for plaque purification. Vectors were constructed with deletions in the E1, E3, and/or E4 regions. We have generated more than 100 vectors with a number of different transgenes and regulatory elements. In addition, the YAP/PAC vector was used to capture a DNA fragment encompassing the human factor IX gene, demonstrating the utility of this system to clone and analyze genomic DNA. This novel cloning strategy allows the rapid and versatile construction of adenoviral vectors for gene expression and gene therapy applications.